Inhibitory Effect of Tetramerized Single-Chain Variable Fragment of Anti-Cyclic Citrullinated Peptide Antibodies on the Proliferation, Activation, and Secretion of Cytokines of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis In Vitro Co-Culture System

Abstract

Tetramerized single-chain variable fragment (ScFv) of anti-cyclic citrullinated peptide (TeAb-CCP) is a constructed tetramerized ScFv of anti-cyclic citrullinated peptide (CCP) antibodies with p53 tetrameric domain, aim to investigate its effect on fibroblast-like synoviocytes (FLSs) proliferation, migration, invasion, and production of inflammatory mediators in the in vitro co-culture system of peripheral mononuclear cells (PBMCs) and FLSs. TeAb-CCP was constructed by modifying a monovalent ScFv antibody to CCP with p53 tetrameric domain to improve its affinity. FLSs were isolated and cultured from rheumatoid arthritis (RA) patients and control subjects. A co-culture system of peripheral mononuclear cells (PBMCs) and FLSs was used. FLSs proliferation, migration, and invasion were measured by MTT, scratch test, and Transwell chamber. Supernatants were measured for cytokines, chemokines, metalloproteinases, and anti-CCP antibodies by Luminex liquid phase protein chip and ELISA. TeAb-CCP significantly inhibited FLSs proliferation in a dose-dependent mode, with maximal action at concentration of 100μg/ml on the 7th day in the co-culture system with PBMCs and FLSs, but not the same with only FLSs. TeAb-CCP significantly suppressed FLSs migration and invasive ability compared with the controls. Significantly lower levels of interleukin (IL)-6, IL-8, RANKL, protein arginine deiminase (PAD)-2, PAD4, metalloproteinase (MMP)-1 and MMP-3 and anti-CCP antibodies were found in co-culture supernatant of TeAb-CCP group. In contrast, transforming growth factor-β (TGF-β) and tissue inhibitor of metalloproteinases-2 (TIMP-2) was significantly increased in the TeAb-CCP group. No significant difference of IL-1a, IL-10, IL-17, TNFα, VEGF, and FGF was found between two groups. As a blocking antibody, TeAb-CCP can significantly inhibit PBMCs of RA to produce pro-inflammatory mediators, and furthermore, inhibit the proliferation, activation, migration, and invasion of FLSs in vitro. In turn, it is suggested that citrullinated modified self-epitopes may be a new target for RA therapy.